Method for detecting specific nucleic acid sequences

ABSTRACT

The present invention relates to a method and test kit for detecting specific nucleic acid sequences, comprising the steps of: 1. matrix-dependent new synthesis of the target nucleic acid; 2. target-specific probe hybridization; and 3. detection of the hybridization event. The invention is characterized in that, in the first step, an oligonucleotide 1, which is marked by a marker 1 and is entirely or partially complementary to the target sequence, acts as a primer in the matrix-dependent new synthesis of the target nucleic acid and, in the second step, an oligonucleotide 2, which is marked by a marker 2 and, owing to its melting temperature being lower than that of the oligonucleotide 1, is not involved in the first step, partially or completely hybridizes with the DNA new synthesis product of oligonucleotide 1.The detection of the hybridization reaction can take place both fluorometrically in the form of a homogeneous assay and, for verification of the result, subsequently immunologically. The detection reaction always takes place in time after the matrix-dependent new synthesis has been carried out.

The present invention relates to a method and a test kit for detectingspecific nucleic acid sequences, comprising the steps of 1.matrix-dependent de novo synthesis of the target nucleic acid, 2.target-specific probe hybridization and 3. detection of thehybridization event. In this context the detection of the hybridizationreaction may be accomplished both fluorimetrically in the form of ahomogeneous assay and thereafter immunologically for verification of theresult. The detection reaction always takes place chronologically afterthe matrix-dependent de novo synthesis has ended.

PRIOR ART

Gene diagnostics have become an indispensable part of modern medicallaboratory diagnostics, forensic diagnostics, veterinary medicallaboratory diagnostics or food and environmental diagnostics.

Genetic diagnostics were revolutionized with the invention of the PCRtechnology, which makes it possible to amplify any desired nucleic acidsequence specifically.

Using PCR, there exists a large number of methods which, in combinationwith the PCR technology, also permit the specific detection of acompleted amplification. In particular, to meet the requirements ofexact genetic diagnostics, there must be used techniques that ensurethat a generated amplification product also corresponds to the targetsequence to be specifically detected. The widespread use ofvisualization of a PCR product by means of gel electrophoresis is notsufficient for this purpose.

One option for detecting specific nucleic acids that in principle isvery rapid and can be accomplished without great experimental time andeffort is known as the real-time PCR method. In this case theamplification reaction is coupled directly with the actual detectionreaction.

A widely used method for detecting specific nucleic acids is theLightCycler technology of Roche. For this purpose the Roche Companydeveloped special hybridization probes, consisting of two differentoligonucleotides, each of which is labeled with only one fluorophore.The acceptor is located at the 3′-end of the one probe, while the otheroligonucleotide has a donor at the 5′-end. The probes are chosen suchthat they both bind to the same DNA strand, wherein the distance betweenacceptor and donor is permitted to be only 1 to 5 nucleotides at most,so that the so-called FRET effect can develop. The fluorescence ismeasured during the annealing step, in which light of this wavelength isdetectable only as long as both probes are bound to the DNA. In thissystem the melting points of both probes should be identical. By virtueof the use of two hybridizing probes in addition to the primers used,the specificity of this detection system is extremely high.

A further real-time PCR application for detection of specific nucleicacid targets can be performed with so-called double-dye probes, whichwere disclosed in U.S. Pat. Nos. 5,210,015 and 5,487,972 (TaqManprobes). Double-dye probes carry two fluorophores on one probe. In thiscase the reporter dye is located at the 5′-end and the quencher dye atthe 3′-end. In addition, a phosphate group may also be located at the3′-end of the probe, so that the probe cannot function as a primerduring elongation. As long as the probe is intact, the released lightintensity is low, since almost the entire light energy produced afterexcitation of the reporter is absorbed and converted due to the spatialproximity of the quencher. The emitted light of the reporter dye is“quenched”, i.e. extinguished. This FRET effect continues to exist afterthe probe has bound to the complementary DNA strand. During theelongation phase, the polymerase encounters the probe and hydrolyzes it.The ability of the polymerase to hydrolyze an oligonucleotide (or aprobe) during strand synthesis is known as 5′-3′ exonuclease activity.Not all polymerases have 5′-3′ exonuclease activity (Taq and Tthpolymerase). This principle was described for the first time for Taqpolymerase. The principle is known as the TaqMan principle. After probehydrolysis, the reporter dye is no longer in the spatial proximity ofthe quencher. The emitted fluorescence is now no longer converted andthis fluorescence increase is measured.

Further methods based on the FRET effect are also known, which methodsdetect both the decrease and the increase of the sample fluorescence.

In the publication JP 002001029072 AA, reporter and quencher are coupledon the individual dNTPs (deoxyribonucleotides) added to the reaction.When these nucleotides are incorporated into the amplified nucleic acid,the fluorescence of the sample is reduced by the FRET effect. Adisadvantage of the method is the incorporation of the labelednucleotides even in the case of mispriming and of primer dimers. Thussuch a method cannot be used for diagnostic applications.

Further publications relate to methods in which the FRET pair labels arenot attached to one oligonucleotide but instead are distributed overseveral primer or probe molecules.

In unexamined application WO 2009/126678 A2, two hybridization probesform a hairpin formation, by which the reporter and quencher approachone another spatially.

In unexamined application DE 10250948 A1, the respectively labeledprobes are hybridized with one another. When a target nucleic acid ispresent, one of the probes is able to form a duplex with the targetnucleic acid, whereby the FRET interaction is canceled.

In unexamined application DE 102005000021 A1, two labeled probes form atriplex with the target nucleic acid, wherein the fluorescence isreleased by the exonuclease activity of the Taq polymerase.

In the patent EP 1384789 B1 and in a publication cited in the patent(Bernard, P. S., at al. Anal Biochem 255 (1998) 101-7), one labeledprimer and one labeled probe respectively are used for real-timeobservation of the amplification.

A further option for specific detection of amplification products bymeans of real-time PCR technology consists in the use of intercalatingdyes (ethidium bromide, Hoechst 33258, Yo-Pro-1 or SYBR Green™, etc.).After excitation by high-energy UV light, these dyes emit light in thevisible lower-energy wavelength range (fluorescence). If the dye ispresent as a free dye in the reaction mixture, the emission is veryweak. It is only by the intercalation of the dye, i.e. by incorporationinto the small furrows of double-strand DNA molecules, that the lightemission is greatly intensified. The dyes are inexpensive anduniversally usable, since in principle any PCR reaction can be followedin real time with them. Moreover, they have a high signal strength,since every DNA molecule is able to bind several dye molecules.Nevertheless, an extreme disadvantage for application also results fromthe advantages: In principle, it is not possible with intercalating dyesto distinguish between correct product and amplification artifacts (suchas primer dimers or nonconforming products). Formed primer dimers andother artifacts naturally also bind intercalating dyes and thereforelead to an unspecific increase of fluorescence even in negative samples.However, a clear differentiation between a specific amplification eventand an artifact is absolutely necessary. In order to achieve this in anycase, there is used a so-called melting-point analysis at the end of theactual PCR reaction. For this purpose the reaction mixture is heated insteps of 1 degree from 50° C. to 95° C. During this process thefluorescence is measured continuously. The point at which thedouble-strand DNA melts is characterized by a drop (peak) of thefluorescence of the intercalating dye, since the intercalating dyedissociates from the single-strand DNA. When the PCR is optimallyadjusted, a sharply accentuated melting-point peak should be expected.This melting point represents the specific product to be expected.Products of different sizes and products from different sequences havedifferent melting points.

Furthermore, by means of real-time PCR applications it is also possibleto achieve quantification of the target to be detected.

As already explained, the described methods satisfy the requirement ofspecific detection of an amplification product.

Nevertheless, a major disadvantage exists in the fact that they areimplemented on very expensive instrumental platforms, which must combinethe processes of both amplification and of subsequent optical detectionappropriate for the problem in one hardware solution. Furthermore, manyof these described detection methods are still based on real-timetracking of the amplification process. On the basis of this strategy,the processing of the measured fluorescence values also takes place inthe course of the amplification reaction. It is clear to the personskilled in the art that an enormously high level of analysis algorithmsmust therefore be integrated into real-time systems. This ultimatelyexplains the large financial expense that must be invested for the useof real-time PCR systems. Finally, even the operation of suchinstrumental systems necessitates a high level of expertise.

Besides the described diagnostic detection methods based on real-timePCR, however, alternative variants also exist for specific detection ofnucleic acids.

In this connection, less expensive methods for detecting nucleic acidsinclude, for example, PCR ELISA. In this method the DNA sequence to beinvestigated is amplified and the produced DNA fragment is thencovalently immobilized on a solid phase (e.g. microtiter plate orstrip), subsequently denatured to a single strand and hybridized with asequence-specific probe. The successful binding of the probe can bevisualized by an antibody-mediated color reaction. Another variant isbased on immobilizing the probe on a solid phase and then bringing thePCR product after the end of denaturing into contact with theimmobilized probe. The detection of a completed hybridization eventtakes place by analogy with the first method variant.

In principle, PCR ELISA techniques are simple to perform, butnevertheless comprise multiple process steps, so that several hours ofworking time to perform the subsequent detection method are also neededin addition to the time needed to perform the PCR. Such a method usuallyneeds 8 hours and therefore is also not suitable as a rapid test.

Furthermore, some instruments such as a temperature-regulating station,a so-called washer or even a measuring instrument for detection of thehybridization signal are also necessary. In addition, further specialinstruments or special consumables may be necessary.

Further simple methods for detection of amplification products are basedon amplification of the target sequences and subsequent hybridization ofamplification products on a membrane. Even in these methods, severalvariants known to the person skilled in the art exist. Once again,however, these methods are laborious to perform, need a large number ofprocess steps to be executed and are therefore not suitable as rapidtests. This then includes even the use of biochip strategies, whichutilize the hybridization of PCR products with hybridization probes todetect the specificity. These methods are also complex and dependent onvery expensive instrumental platforms.

A significant reduction of working steps is disclosed in the publicationKR 1020060099022 A (Method and kit for rapid and accurate detection andanalysis of nucleotide sequence with naked eye by using membrane lateralflow analysis).

In this case a so-called lateral-flow method is used to detect nucleicacids. This method also depends on the technology of hybridization ofnucleic acids on a solid phase. One advantage of lateral flow methods isthat they represent a small, manual test format (strip test).

A very rapid detection method that also depends on the detection ofamplification products by means of a test strip and is commerciallyavailable is in turn based on a completely different principle from thatin the above publication. In this case, the PCR reaction is performedwith one biotinylated primer and one non-biotinylated primer. Aftercompletion of the PCR, a PCR product labeled with biotin at one end istherefore obtained. A test strip (e.g. of Millenia, Amodia, etc.)containing two separate binding sites is used for detection: astreptavidin site for coupling the biotin-labeled DNA strand and an FITCbinding site for checking the function of the test strip.

The PCR product is detected by denaturing the PCR batch at the end ofthe PCR and hybridizing with a probe complementary to the biotin-labeledDNA strand. The probe is FITC-labeled.

For detection, the PCR hybridization batch is mixed with a run bufferand applied on the test strip. According to the description of the test,the biotinylated DNA strand binds to the streptavidin binding site ofthe strip. Detection is accomplished via the FITC label of the probehybridized with the DNA strand. A typical signal in the form of a stripeappears. This signal is supposedly the specific detection of theamplification product. However, the method does not combine thehybridization of the probe with the PCR process but instead performs itas a separate method step.

Unexamined application WO 2009/000764 A2 permits the performance ofamplification and hybridization of the PCR product in one reactionbatch. In this case there is formed a doubly labeled amplificate-probedimer, which can then be visualized, e.g. by means of a lateral-flowstrip. This is a very inexpensive way of visualizing a PCR hybridizationproduct, and in particular is independent of instrumentation. Adisadvantage of this method, however, is that primarily it is not ahomogeneous assay, since the reaction vessel must be opened after theamplification/hybridization reaction has taken place, in order that theamplification mixture can be transferred to a lateral-flow strip.

Thus the actual detection reaction takes place outside the reactioncavity in which the amplification/hybridization reaction is performed.

Also, the method does not permit any numerical display of the detectionresult, but is always limited merely to a YES-NO decision based onvisual observation. Quantification of the reaction products is also notpossible on a lateral-flow strip, since the upper limit is dictated bythe binding capacity of the strip.

In International Patent Application WO 2005/51967 A2, labeledoligonucleotides with several fluorophores are described. The method forseparating the fluorophores, including cleaving the labeledoligonucleotides, uses enzymes with 5′-exonuclease activity.

The subject matter of the publication WO 03/072051 A2 is a fluorescenceenergy transfer (FET) labeled probe with a nucleic acid intercalator,which contains a polycyclic compound bound to an FET-labeledoligonucleotide, wherein the nucleic acid intercalator is covalentlybound at the 3′-end of the FET-labeled oligonucleotide, and wherein theFET-labeled oligonucleotide represents a dark quencher, which ispositioned at its 3′-end, and wherein the FET-labeled probe is resistantto 3′-5′-exonuclease.

The publication of E Lyon et al., “LightCycler Technology in MolecularDiagnostics”, J. Mol. Diagn (2009) 11 (2) 93-101, reports on a PCR withreal-time fluorescence monitoring and melting curve analysis. Thispermits the PCR to be performed within 15 minutes. The review describesthe significant advances of LightCycler technology within the last 15years.

The objective of the present invention is to provide a universallyusable method for specific detection of target nucleic acids, whichmethod also makes it possible to perform the detection reaction in theform of a homogeneous assay, meaning that the detection of the detectionreaction already takes place in the reaction cavity, in which the actualamplification/hybridization reaction is also occurring. Anotherobjective was to permit diagnostic certainty of a detection reaction byquasi dual detection, since a series of test procedures necessitates notonly a first detection reaction (such as real-time PCR) but also asecond control detection (such as application of the amplificationproducts on an agarose gel). As an expansion of the objective, such anovel method could also be used in such a way that the detectionreaction takes place as a homogeneous assay (instrument-dependent) orelse, as an alternative thereto, even as a test based on a lateral-flowstrip (instrument-independent).

A method with a similar objective was published by Piepenburg et al.(PLOS biology, July 2006, Volume 4, Issue 7, 1115-1121). In this case arecombinase-coupled isothermal PCR is performed. The double-labeledprobe has an integrated cut site for a nuclease. During hybridization ofthe probe, the formed double strand is cut by the nuclease and therebythe fluorescence is released. After having been cut with the nuclease,the labeled probe is able to function as a primer. Together with theother labeled primer, there is then formed a double-labeled PCR product,which can be detected on a LFA strip. One disadvantage is that thismethod is chemically very complex and difficult. Furthermore, evenbefore the beginning of the test procedure, it must be decided which ofthe detection methods and therefore which labels are preferred. If sucha decision were not made, then the probe must be prepared at a minimumof four sites: 1. reporter fluorophore, 2. quencher, 3. cut site of thenuclease, 4. amplification blockade at the 3′-end.

The present object was solved surprisingly simply according to thefeatures of the claims. The inventive method combines a matrix-dependentDNA de novo synthesis with a hybridization step. The inventive choice oflabels of the oligonucleotides participating in the reactions permitsnot only a reaction-dependent fluorescence measurement and associatedtherewith a numerical and possibly quantitative evaluation but alsoinstrument-independent visualization of the reaction, e.g. on alateral-flow strip. It is also particularly advantageous that thedetection of the fluorescence reduction following a FRET effect ispossible with the inventive method in the form of end-point detection.From the instrumental viewpoint, such a measurement principle cantherefore also dispense with the use of expensive real-time instrumentalsystems.

This inventive method is based on the following steps:

A. Supplying a Reaction Batch, Consisting of:

-   -   a sample containing a nucleic acid, in which the target nucleic        acid is to be detected    -   at least one oligonucleotide labeled with a label 1, which is        completely or partly complementary to the target sequence and        functions as a primer in a matrix-dependent de novo synthesis of        the target nucleic acid (oligo type 1)    -   at least one oligonucleotide labeled with a label 2, which by        virtue of the lower melting temperature than that of        oligonucleotide 1 does not participate in the DNA de novo        synthesis process, but is able to hybridize partly or completely        with the DNA de novo synthesis product of oligonucleotide 1        (oligo type 2)    -   a mixture of chemicals/enzymes, possibly also with further        unlabeled oligonucleotides, for permitting a matrix-dependent de        novo synthesis of the target nucleic acid.

As used in the invention, the term “partly complementary” means thatsufficient complementarity must be present. In the present case, atleast 50%, preferably 70% of the labeled oligonucleotide must becomplementary to the target nucleic acid.

As used in the invention, the term “matrix-dependent” means that the denovo synthesis of the target nucleic acid is controlled by the primersbeing used.

According to the invention, the labels of the two oligonucleotides(oligo type 1 and oligo type 2) are chosen such that together they forma FRET pair (such as FITC/TAMRA, FAM/TAMRA, FAM/BHQ1, etc.) and, inrelation to the inventive dual detection, are also capable of havingcomplementary binding partners on a lateral-flow strip.

B. Performing the Matrix-Dependent DNA De Novo Synthesis with IntegratedProbe Hybridization

Depending on the type of target nucleic acid, there is performed eithera reverse transcriptase reaction (in the case of RNA, which occurs in avery large number of copies, such as rRNA, tmRNA) or amplification (inthe case of DNA); it is even possible (in the case of a rare RNA, suchas mRNA, samples with a small number of particles) to perform the tworeactions in succession.

By virtue of the inventive method, only oligo type 1 participates inthis first reaction. The oligo type 1 functions as a primer either in anRNA-dependent reverse transcription (whereby a labeled cDNA strand isformed) or in amplification of the target DNA or cDNA (whereby a labeledPCR product is formed). One-step RT-PCR can also be performed. In thisprocess a second unlabeled primer oligonucleotide increases the yield ofthe PCR reaction.

By virtue of its lower annealing temperature in accordance with theinventive method, the oligo type 2 does not participate in the DNA denovo synthesis. Thereafter the reaction batch is heated to a temperatureof >90° C. This step leads to thermal separation of the strands. Afterthe end of this thermal denaturing reaction, the reaction batch iscooled to the hybridization temperature of the oligo type 2. During thisstep, the oligo type 2 binds specifically to the complementary DNAstrand. This strand then carries label 1, which was incorporated intothe reaction product by the oligo type 1.

C. Detection of the Hybridization Event

The detection reaction can take place in two variants, but according tothe invention the two detection variants may also be used in parallel orelse may even be combined as a verification reaction.

1. Detection of the Hybridization Reaction by Means of a FluorescenceMeasurement.

The labels incorporated by the two oligos type 1 and type 2 form a FRETpair. The hybridization of the oligo type 2 with the synthesis productof the oligo type 1 that takes place in the inventive method leads to aFRET effect between labels 1 and 2. This effect now leads to ameasurable decrease of the fluorescence. This reduction of thefluorescence is numerically evaluated, thus permitting unambiguousdetection of the reaction.

2. Detection of the Hybridization Reaction Outside or Inside theReaction Vessel on a Solid Phase, Characterized in That:

The solid phase (e.g. a lateral-flow strip, microtiter plate,microparticle) contains a binding site for one of the labels of oligotype 1 or type 2 and/or antibodies or other binding molecules againstthe labeling molecules of oligos type 1 or type 2 that are able to bindto the labeling molecules of type 1 or type 2 (for example, covalentbonds or hydrogen bonds or via bridging molecules). Furthermore, adetection molecule for visualization or measurement of the hybridizationevent is located on the solid phase, or such a detection molecule isadded to the detection reaction. However, it is also possible toincorporate the detection molecule into the hybridization product to bedetected as early as during the amplification/hybridization reaction.

In summary, an extremely simple and universally usable detection methodfor gene diagnostics is now available with the inventive method.

According to the invention, the detection of a diagnostically relevanttarget nucleic acid to be detected takes place in the form of ahomogeneous assay via the end-point fluorescence measurement offluorescence quenching. The result can be acquired numerically and italso permits quantification of the target nucleic acid to be detected(using an internal standard) detected and quantified. Furthermore, theinventive method also permits highly specific dual detection, sinceafter fluorescence detection has been achieved the result can beverified on a lateral-flow strip. From the diagnostic viewpoint, such averification is therefore very much more exact than the detection thathas been possible heretofore of real-time PCR products on an agarosegel. If necessary, the method also makes it possible to perform thetests independently of one another (test by means of fluorescencedetection or test by means of detection on, for example, a lateral-flowstrip).

This elegant novel test procedure, and especially also the combined testprocedure (fluorescence detection followed by verification of the firsttest reaction on a solid phase), are made possible according to theinvention by the fact that the hybridized probe (oligo type 2) is notdecomposed by the Taq polymerase during amplification/hybridization butinstead remains in the hybridized condition even after the end of thereaction, in contrast to the homogeneous TaqMan exonuclease assay.

The inventive integration of a hybridization probe into the reactionprovides the certainty that the amplified fragment actually contains thetarget sequence. Thereby false-positive results caused by mispriming areexcluded. The use of the chemically modified probe (preferablyphosphorylation of the last nucleotide of the probe) prevents theextension of the probe by 5′→3′ polymerase activity and thus preventsthe probe from functioning as a primer and generating unspecific PCRartifacts (primer dimers), which would be detected as false-positivesignals.

In contrast to real-time PCR methods, the detection of the specificdetection signal takes place not during amplification, where thefluorescence is released either due to the probe hydrolysis caused bythe Taq polymerase (EP 0972848 A2) or is reduced by the FRET effect (EP1384789 B1), but only after the end of the amplification-hybridizationreaction. This also causes the positive effect that the method isindependent of instrumental equipment. Measurements may be made both ina real-time PCR instrument and after the end of the reaction with afluorescence reader (see exemplary embodiments).

The inventive method also differs from the patent (EP 0826066 B1) thatalso describes a combination of PCR and hybridization. In this methodalso, a FRET-effect-mediated fluorescence signal is again detected. Thisoccurs during the process of amplification by hybridization of a doublylabeled probe having a lower annealing temperature than that of theprimer. The release of fluorescence in this case takes place not byhydrolysis of the probe as a result of the exonuclease activity of thepolymerase but instead by the fact that the secondary structure of theprobe is loosened during hybridization and fluorescence is released bythe increase of the distance of the reporter from the quencher. In thiscase only enzymes having no exonuclease activity (e.g. Klenow fragmentsor T4 or T7 polymerases) can be used for amplification.

For the first time there has been achieved a homogeneous method fordetecting the presence of a target nucleic acid in a sample, wherein thereaction batch contains:

-   -   a sample nucleic acid, in which the target nucleic acid is        suspected    -   at least one oligonucleotide, which is labeled by a label 1, is        completely or partly complementary to the target sequence and        functions as a primer in a matrix-dependent de novo synthesis of        the target nucleic acid (oligo type 1)    -   at least one oligonucleotide labeled with a label 2, which by        virtue of the lower melting temperature than that of        oligonucleotide 1 does not participate in the DNA de novo        synthesis process, but is able to hybridize partly or completely        with the DNA de novo synthesis product of oligo type 1 (oligo        type 2)    -   a mixture of chemicals/enzymes, possibly also with further        unlabeled oligonucleotides, for permitting a matrix-dependent de        novo synthesis of the target nucleic acid.

The method comprises the following steps:

-   -   matrix-dependent de novo synthesis of the target nucleic acid to        be detected with at least one oligo of type 1 and possibly        subsequent strand separation    -   hybridization of the synthesis product of the respective oligo        type 1 with at least one oligo type 2    -   detection of the hybridization reaction by means of a        fluorescence measurement.

Labels 1 and 2 form a FRET pair. The hybridization of the oligo type 2with the synthesis product of the oligo type 1 leads to a measurabledecrease of the fluorescence caused by the FRET effect between labels 1and 2.

Detection of the hybridization reaction is possible outside or insidethe reaction vessel. In the case of detection on a solid phase, thesolid phase contains a binding site for one of the labels of the oligotype 1 or type 2 and/or antibodies or other binding molecules againstthe labeling molecules of oligos type 1 or type 2, which bind to thelabeling molecules of type 1 or type 2 (for example, covalent bonds orhydrogen bonds or via bridging molecules) and at the same time adetection molecule for visualization or measurement of the hybridizationevent, or such a molecule is added to the sample bound to the solidphase.

Oligos type 1 and type 2 may also carry labels other than the labelsnamed in claim 4. The additional labels may be used for detection of thehybridization event on the solid phase (1e).

It is also possible to perform an asymmetric amplification instead ofthe standard amplification.

According to a preferred embodiment of the invention, the meltingtemperature (T_(m)) of oligo type 1 is preferably 5° C. to 15° C. higherthan the T_(m) of oligo type 2. After hybridization, labels 1 and 2 arepreferably 1 to 50 by apart from one another.

It is also possible for the oligo with a reporter label to be present inthe reaction in a lower concentration than the oligo with the quencherlabel, preferably in the ratio of 1:10 to 1:20.

The inventive method will be described hereinafter on the basis ofexemplary embodiments, but the exemplary embodiments are not to beconstrued as any limitation of the method.

EXEMPLARY EMBODIMENTS Example 1

Detection of Influenza Type H1N1 (Swine Origin cDNA) by Means ofHybridization Methods Integrated into the PCR and End-Point FluorescenceMeasurement of Fluorescence Quenching

Negative samples (NTC), H1N1 cDNA-positive samples (POS) and the samplesthat indeed contained human DNA material (swab smear of nasal mucousmembranes) but not H1N1 (NEG) were present in the batch.

The possibility of an inventive end-point fluorescence measurement andthe specificity of the method will be demonstrated with this example.

PCR Primer/Probe

H1N1 sense primer (5′-tgg gaa atc cag agt gtg aat cac tct c-3′)H1N1 antisense primer (oligo type 1)(5′-BHQ1-cgt tcc att gtc tga act agr tgt ttc c-3′)H1N1 probe (oligo type 2) (5′-agc aag ctc atg gtc cta cat t-FAM-3′)

Samples: 3× POS; 3× NEG; 3× NTC Per Sample:

sense primer (25 pmol/μL) 0.1 μL antisense primer (50 pmol/μL) 0.1 μLprobe (5 pmol/μL) 0.1 μL dNTP mix (12.5 mM) 0.3 μL 10X PCR buffer (MgCl₂included) 1.5 μL Taq-DNA polymerase 0.75 U PCR grade H₂O add 15 μL

The PCR was carried out in the SpeedCycler (Analytik Jena) using therapid cycler technology:

Amplification/Hybridization Conditions

Step 1: denaturing 98° C./90 sec Step 2: amplification for 41 cycles(98° C./4 sec; 57° C./4 sec; 72° C./10 sec) Step 3: denaturing 95°C./900 sec Step 4: hybridization: 43° C./600 sec

The amplification event/the hybridization reaction was detected by meansof an end-point measurement with the SpeedScan fluorescence reader(Analytik Jena AG; FIG. 2). At the same time, the change of fluorescenceintensity of the sample was measured in real time during the entirereaction (FIG. 3). The measured data of the SpeedScan instrument weresubjected to a Pos/Neg determination according to the following formula:

xN−xN _(min)+% xN=A

xN−P=B

if A−B≧0, the sample is negative

if A−B<0, the sample is positive

Where xN is the mean value of the NTC values; xN_(min) is the smallestNTC value; desired percentage deviation of the positive value from thenegative value (e.g. 20% of xN), and P is the measured value of thesample to be tested.

Furthermore, the sample may be evaluated semi-quantitatively by using aconcentration standard.

Nevertheless, a truly quantitative evaluation is possible only in thepresence of an internal control and a competitive reaction.

The results of the qualitative evaluation of the sample fluorescence aresummarized in Table 1.

Measured value Value A at Sample ID after the PCR 20% Value B A − Bpos/neg Pos 3447 11485 15506 −4021 POS Pos 6533 11485 12420 −935 POS Pos6024 11485 12929 −1444 POS Neg 18408 11485 5573 5912 NEG Neg 11764 114857189 4296 NEG Neg 18937 11485 16 11469 NEG NTC 11258 11485 7695 3790 NEGNTC 26666 11485 −7713 19198 NEG NTC 18937 11485 16 11469 NEG

Example 2

Dependence of Signal Intensity on the Concentration of the Target DNA inComparison with a Conventional Real-Time PCR

Two batches were prepared: for the inventive method and a real-time PCRbatch with a probe labeled with FAM-BHQ1. The cDNAs (see table forparticle count/PCR batch) synthesized from Influenza H1N1 virus strainswere used as samples.

-   Batch 1: See Example 1 for the reaction conditions-   Batch 2:

PCR Primer/Probe

H1N1 RT sense primer (5′-tgg gaa atc cag agt gtg aat cac t-c-3′)H1N1 RT antisense primer (5′- cgt tcc att gtc tga act agr tgt t-3′)H1N1 RT probe (5′-FAM-cca caa tgt agg acc atg agc ttg ctg t-BHQ1-3′)

Per Sample:

sense primer (50 pmol/μL) 0.1 μL antisense primer (50 pmol/μL) 0.1 μLprobe (25 pmol/μL) 0.1 μL dNTP mix (12.5 mM) 0.3 μL 10X PCR buffer(MgCl₂ included) 1.5 μL Taq-DNA polymerase 0.75 U PCR grade H₂O add 15μL

The PCR was carried out in the SpeedCycler (Analytik Jena) using therapid cycler technology:

Amplification/Hybridization Conditions

Step 1: denaturing 98° C./90 sec Step 2: amplification 41 cycles (98°C./4 sec; 57° C./4 sec; 72° C./10 sec)

The amplification event/the hybridization reaction in Batch 1 wasdetected by means of an end-point measurement of the fluorescence bymeans of SpeedScan (Analytik Jena AG; FIG. 4). At the same time, thechange of fluorescence intensity of the sample was observed in real time(real-time PCR) during the entire reaction (FIG. 5). The measured dataof the SpeedScan instrument were subjected to a Pos/Neg determinationaccording to the formula described hereinabove (see Example 1). In Batch2, the fluorescence release was measured conventionally by means ofreal-time PCR.

The results of the qualitative evaluation of the sample fluorescence aresummarized in Table 2.

Particle Measured count value after in the the PCR Value A Ct valuesample (mean value) at 20% Value B A − B pos/neg of Batch 2 10000 75474910 7292 −2382 POS 26 5000 3650 4910 11189 −6279 POS 26 500 9400 49105439 −529 POS 33 50 10816 4910 4023 887 NEG 38 5 10590 4910 4249 661 NEGNoCt NTC 14839 4910 0 4910 NEG NoCt

EXPLANATION OF THE FIGURES

FIG. 1 shows a diagram of the process flow.

FIG. 2 shows a fluorescence measurement after the end of theamplification/hybridization reaction. Fields B3-B5 are POS samples,B6-B8 are NEG samples, B9-B11 are NTC samples (measurement of thefluorescence by means of SpeedScan (Analytik Jena AG)).

FIG. 3 shows a real-time fluorescence measurement during the entireamplification/hybridization reaction. Curves 1-3 are POS samples, 4-6are NEG samples, 7-9 are NTC samples (real-time measurement for trackingof the detection reaction).

FIG. 4 shows a fluorescence measurement after the end of theamplification/hybridization reaction. Fields B3/C3 are POS samples witha virus particle count of 10000; B4/C4 are POS samples with a virusparticle count of 5000; B5/C5 are POS samples with a virus particlecount of 500; B6/C6 are POS samples with a virus particle count of 50;B74/C7 are POS samples with a virus particle count of 5; B9-B10/C9-C10are NTC samples.

FIG. 5 shows a real-time fluorescence measurement during the entireamplification/hybridization reaction. Curves 1-4 are NTC samples;samples 5-14 are concentrated as follows: 5, 6-100000 particles/sample;7, 8-5000 particles/sample; 9, 10-500 particles/sample; sample 11, 12-50particles/sample; sample 13, 14-5 particles/sample.

FIG. 6 shows a real-time fluorescence measurement during a real-timereaction with a conventional TaqMan probe. Curves 6, 7 are NTC samples;samples 1-5 are concentrated as follows: 1-100000 particles/sample;2-5000 particles/sample; 3-500 particles/sample; sample 4-50particles/sample; sample 5-5 particles/sample.

1. A method for detecting a specific nucleic acid sequence, the methodcomprising: a) synthesizing a DNA target nucleic acid in amatrix-dependent de novo manner, b) hybridizing the target nucleic acidwith a probe, and c) detecting the hybridizing, wherein: a primer in a)comprises an oligonucleotide 1 labeled with a label 1, which iscompletely or partly complementary to the target nucleic acid, and theprobe in b) comprises an oligonucleotide 2 labeled with a label 2, whichhas a lower melting temperature than oligonucleotide 1 and does notparticipate in the synthesizing, but which hybridizes partly orcompletely with the target nucleic acid.
 2. The method of claim 1,wherein the detecting is performed in a reaction cavity in which thesynthesizing and hybridizing are also performed.
 3. The method of claim1, wherein the detecting is performed on a solid phase, either inside oroutside of a reaction cavity in which the synthesizing and hybridizingare performed.
 4. The method of claim 2, wherein the two labels 1 and 2as are a FRET pair, and the detecting comprises measuring a decrease influorescence.
 5. The method to of claim 4, wherein the detectingcomprises end-point detecting.
 6. The method of claim 1, furthercomprising, after a), heating a reaction batch heated to a temperatureof greater than 90° C., and thereafter cooling the reaction batch is toa hybridization temperature of oligonucleotide
 2. 7. The method of claim1, wherein the hybridized oligonucleotide 2 is not destroyed by a Taqpolymerase during the synthesizing or hybridizing, but instead remainsin a hybridized state even after the synthesizing and hybridizing. 8.The method of claim 1, wherein the detecting is performed only after thesynthesis and hybridization are finished.
 9. The method of claim 3,wherein the solid phase comprises: a site that binds to at least oneselected from the group consisting of label 1, label 2, an antibodyagainst label 1, an antibody against label 2, a binding molecule againstlabel 1, and a binding molecule against label 2; and wherein the solidphase comprises a detection molecule that visualizes or measures thehybridizing.
 10. The method of claim 1, wherein oligonucleotide 2 isprotected against a 5′Δ3′-polymerase activity.
 11. A test kit thatperforms the method of claim 1, comprising: the oligonucleotide 1,labeled with the label 1, the oligonucleotide 2 labeled with the label2, and a mixture of chemicals, enzymes, or both, optionally with afurther unlabeled oligonucleotide.
 12. The test kit of claim 11, whereinthe melting temperature of the oligonucleotide 2 is 5° C. to 15° C.lower than that of the oligonucleotide
 1. 13. The method of claim 4,wherein the FRET pair is selected from the group consisting ofFITC/TAMRA, FAM/TAMRA, and FAM/BHQ1.
 14. The method of claim 1, whereinthe oligonucleotide 2 is protected against a 5′→3′-polymerase activityby the label
 2. 15. The method of claim 1, wherein the oligonucleotide 2is protected against a 5′→3′-polymerase activity by phosphorylation. 16.The method of claim 1, wherein the oligonucleotide 1 is at least 50%complementary to the target nucleic acid.
 17. The method of claim 1,wherein the oligonucleotide 1 is at least 70% complementary to thetarget nucleic acid.
 18. The method of claim 1, wherein theoligonucleotide 1 is completely complementary to the target nucleicacid.
 19. The method of claim 1, wherein the melting temperature of theoligonucleotide 2 is 5° C. to 15° C. lower than that of theoligonucleotide
 1. 20. The method of claim 1, wherein after thehybridizing, the labels 1 and 2 are located 1 to 50 base pairs apartfrom each other.